Enhanced absorption of prenylated cinnamic acid derivatives from Brazilian green propolis by turmeric in humans and rats

Abstract Prenylated cinnamic acid derivatives are the bioactive components of Brazilian green propolis (BGP). The effect of other botanical components on the pharmacokinetic profiles of these derivatives remains relatively unexplored. In the present study, we investigated the influence of several herbal extracts (turmeric, ginkgo leaf, coffee fruit, soybean, and gotu kola) on the plasma concentrations of cinnamic acid derivatives after BGP consumption. When the herbal extracts were co‐administered with BGP in the clinical study, the area under the curve (AUC) values of artepillin C and drupanin, the major BGP components in plasma, were significantly increased by 1.7‐ and 1.5‐fold, respectively, compared to those after BGP administration alone. Among the herbal extracts administered to rats, turmeric extract increased the AUC. Furthermore, a bidirectional transport assay suggested that artepillin C and drupanin are substrates of breast cancer resistance protein (BCRP), a drug elimination transporter. These results suggest that curcumin‐containing turmeric extract may increase the plasma concentrations of artepillin C and drupanin via BCRP. Our findings enabled us to estimate the food–herb and herb–herb interactions in vivo in foods and herbal medicines containing cinnamic acid derivatives and prenylated compounds.

Recently, Zhu et al. (2018) and Asama et al. (2021) reported improved cognitive function in elderly people owing to the long-term intake of BGP, with artepillin C suggested as the potential active component.
A mixture of BGP and several herbal extracts (turmeric, ginkgo leaf, coffee fruit, soybean, and gotu kola) has been found to improve cognitive function in the elderly (Asama et al., 2020).Curcumin in turmeric, ginkgolides in ginkgo leaves, phosphatidylserine from soybeans, and chlorogenic acid in coffee fruits have been reported to reduce neuroinflammation and exert neuroprotective effects (De Simone et al., 2003;Liu et al., 2023;Xiong et al., 2023;Yu et al., 2018).Additionally, gotu kola presumably improves memory in humans (Fitriana et al., 2021).
Hence, BGP and these herbal extracts may have a synergistic effect on cognitive function.
Generally, the biological activities and pharmacokinetics of bioactive constituents are influenced by the presence of other constituents in food and herbal plants (Phan et al., 2018;Scholz & Williamson, 2007).Several studies have revealed enhanced absorption of herbal constituents due to the co-administration of several herbs (Sun et al., 2019).Recently, green propolis has been reported in vitro to improve the intestinal membrane permeability of curcuminoids (de Albuquerque et al., 2023), suggesting interaction between components of natural products, thereby affecting their absorption.
Therefore, the absorption of BGP constituents is affected by their interaction with other herbs.In our previous studies, we evaluated the uptake and metabolic processes of cinnamic acid derivatives of BGP in humans and rats (Yamaga et al., 2021(Yamaga et al., , 2022)).In particular, artepillin C and drupanin were the primary constituents detected in the plasma after BGP consumption, and some of the components were metabolized to conjugates and hydroxylated metabolites, such as capillartemisin A and 3,4-dihydroxy-5-prenylcinnamic acid.
Baccharin was hydrolyzed to drupanin and 3-phenylpropionic acid and was not detected in any plasma sample.Notably, few studies have investigated the phytochemicals that affect the absorption of cinnamic acid derivatives.Therefore, the objective of this study was to determine the effects of herbal extracts (turmeric, ginkgo leaf, coffee fruit, soybean, and gotu kola) on the absorption of cinnamic acid derivatives of BGP.Herein, we describe the effect of the interaction between BGP and herbal extracts on the pharmacokinetic profiles of cinnamic acid derivatives after a mixture of BGP and herbal extracts was consumed by humans, and a combination of BGP and each herbal extract was ingested by rats.In addition, we discuss the molecular mechanisms underlying this interaction using a cell-based bidirectional transport assay.

| Materials and chemicals
BGP and natural herbal extracts (turmeric, ginkgo leaf, coffee fruit, soybean, and gotu kola) were obtained as freeze-dried powders from Yamada Bee Company, Inc. (Okayama, Japan).The BGP powder (Lot: LY-008) was standardized to contain 8.0% artepillin C and 0.14% culifolin.Turmeric extract powder was standardized to contain 70% curcumin, 15% desmethoxycurcumin, and 2.5% bis-demethoxycurcumin.Gingko leaf extract powder was standardized to contain 24% total flavonol glycosides and 6% total terpene lactones.Coffee fruit extract powder and soybean extract powder were standardized to contain 40% chlorogenic acid and 70% phosphatidylserine, respectively.For the clinical trial, the extract powders were mixed and encapsulated according to the proportions mentioned previously (Asama et al., 2020): 75 mg of BGP powder; 250 mg of turmeric extract powder; 120 mg of ginkgo leaf extract powder; 100 mg of coffee fruit extract powder; 150 mg of soybean extract powder; and 250 mg of gotu kola extract powder.Authentic artepillin C was procured from Fujifilm Wako Pure Chemical Industries Ltd. (Osaka, Japan).Drupanin, capillartemisin A, and 3,4-dihydroxy-5-prenylcinnamic acid were prepared according to the methods outlined in previous reports (Tani et al., 2010(Tani et al., , 2019)).
Sulfatase from Helix pomatia (type H-1 with a minimum activity of 10,000 units/g solid, including β-glucuronidase with a minimum activity of 300 units/mg solid) was purchased from Sigma-Aldrich Inc.

| In vitro study
The Madin-Darby canine kidney (MDCK II) cell line and MDCK II cells overexpressing multidrug resistance 1 (MDR1) or breast cancer resistance protein (BCRP) were purchased from the Netherlands Cancer Institute (Amsterdam, Netherlands) and SOLVO Biotechnology (Szeged, Hungary), respectively.The cell lines were routinely cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in 5% CO 2 .All cells were seeded in a feeder tray at a density of 3-6 × 10 4 cells/well and grown for 3-4 days.On the day of the transport study, cell monolayers were washed with Hanks' balanced salt solution (HBSS) buffer containing 0.1% BSA (pH 7.4) and incubated at 37°C for 20 min.Artepillin C and drupanin dissolved in DMSO (10 mM) were added to the transport buffer (HBSS containing 10 mM HEPES, 0.2% glucose, 0.1% BSA, pH 7.4) to a final concentration of 10 μM and then loaded into the apical or basolateral chamber.After 120 min of incubation, 150 μL of the aliquots were collected from the donor and receiver chambers and mixed with an equal volume of 50% methanol.After centrifugation at 3000 rpm for 10 min at 4°C, the supernatants were filtered and subjected to LC-MS/MS analysis.

| Animal study
This animal study was approved by the Animal Research Ethics Committee of KAC Co., Ltd. (Kyoto, Japan;approval No. 22-1217).
All rats were handled in accordance with the Animal Care and Use Regulations of KAC Co., Ltd.
Wistar/ST rats (male, 7 weeks old) were purchased from Japan SLC (Shizuoka, Japan).Forty-eight rats were housed in laminar flow cages under the following conditions: temperature 23 ± 2°C; relative humidity 45%-60%; 12 h light/dark cycle (from 7:00 a.m. to 7:00 p.m.); and with ad libitum access to water and food.The rats were quarantined and acclimatized under the above conditions for 1 week and divided into six groups (eight rats/group) according to their average body weight (240-260 g).Each test sample listed in Table 1 was dissolved in 10% propylene glycol (1 mL per rat) and administered intragastrically to the rats after an overnight fasting period with free access to water.The dosage ratios of the test samples were determined based on the proportions shown to improve cognitive function in a previous study (Asama et al., 2020).Blood samples were obtained from the tail vein of rats using heparin-coated glass capillaries (Thermo Scientific, Waltham, MA, USA) before the test component administration as well as at intervals of 30 min and 1, 1.5, 2, 3, 6, 9, and 24 h after the administration.The collected samples were centrifuged at 1500 g for 10 min at 4°C to separate the plasma and preserved at −80°C until further sample preparation.

| Clinical trial
The protocol for the clinical trial was reviewed and approved by the Yamada Bee Co. Ethical Committee.This study adhered to the ethical tenets of the Declaration of Helsinki (approval no.2020015).All participants were informed of the research protocol, and they have signed an informed consent form.
Six volunteers (three males and three females) completely understood the study protocol and consented to participate in the research.The following exclusion criteria were used for shortlisting participants: past or present medical history (asthma, significant allergies, heart, liver, kidney, and urinary tract disease); prescribed medication; pregnancy; smoking (>20 cigarettes/day); alcohol consumption (>500 mL/day of beer); use of dietary supplements and healthy foods; and participation in other clinical trials.The participants were instructed to refrain from consuming propolis and propolis-containing foods and beverages during the study period and 1 week prior to the start of the study.After a 12-h fast, the participants were given a test food comprising the encapsulated BGP powder or a mixture of the BGP powder and herbal extracts in two experimental periods separated by 1 week.Blood samples were collected before the ingestion of test food and at 30 min and 1, 1.5, 3, 6, 9, and 24 h after the ingestion.The collected samples were centrifuged at 1500 g for 3 min at 23 ± 2°C to separate the plasma, which was subsequently preserved at −80°C until further sample preparation.

| Plasma sample preparation
Human and rat plasma samples were prepared as previously described (Yamaga et al., 2021(Yamaga et al., , 2022)).Briefly, 50 μL of plasma was treated with a sulfatase/β-glucuronidase solution (sulfatase 15 units/sample) in 100 mM sodium acetate and 5 mM ascorbate at 37°C for 1 h.Following the incubation, 850 μL of methanol was added to the samples to stop the deconjugation reaction, after which centrifugation was performed at 2000 g for 10 min at 23 ± 2°C.The supernatant was evaporated under nitrogen gas and then reconstituted in a fixed volume of methanol.The samples were filtered through a 0.22 μm PTFE membrane and then analyzed by LC-MS/MS.
The elution was performed using solvent A (0.1% formic acid) and solvent B (acetonitrile) at a flow rate of 0.3 mL/min.The gradient profiles were as follows: 5% B (0-2 min), 5-95% B (2-22 min), 95% B (22-25 min), and 5% B (25-30 min).Tandem mass spectrometry (MS/MS) was conducted using an ion trap mass spectrometer with a heated electrospray ion source (HESI) in both positive and negative ionization modes (scan range m/z 70-1000).The mass detection parameters were the same as previously described (Tani et al., 2023).External standard curves for cinnamic acid derivatives TA B L E 1 Combination and dosage of Brazilian green propolis (BGP) and each herbal extract administered to rats.

| Pharmacokinetic analysis
Quantification of cinnamic acid derivatives in the plasma was performed using the Trace Finder software version 4.1 (Thermo Scientific).The plasma concentration versus time data for the analytes obtained from each human and rat were entered into a Microsoft Excel spreadsheet.The area under the plasma concentration-time curve (AUC) was calculated using the linear trapezoidal rule, extending to the last sampling point.

| Data analysis
Plasma concentration data were expressed as mean ± standard deviation, and statistical analyses were performed using GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA).In the clinical study, differences in the plasma concentrations and AUC values of the cinnamic acid derivatives due to the intake of the test foods were analyzed using the paired t-test.For the animal study, two-group comparisons were carried out using Student's t-test, while multiple comparisons were conducted using one-way analysis of variance (ANOVA) followed by Dunnett's post hoc test.Significant differences in the measured variables were recognized when the derived p-value was ≤.05.
The apparent permeability coefficients (Papp) for both directions, from the apical side to the basolateral side (A to B) and from the basolateral side to the apical side (B to A) in the cell monolayer, were calculated using the following equation: where dQ/dt is the permeation rate (nmol/s), A is the membrane area (cm 2 ), and C0 is the initial concentration of the compound in the donor compartment (nM).The efflux ratios (ER) in the B-to-A and A-to-B directions were determined using the following equation: Net flux ratios were calculated by dividing the ER obtained from MDCK II-MDR1 (or BCRP) cells by that obtained from the parent MDCK II cells.A net flux ratio greater than 2 indicated that the efflux of the compound was specifically mediated by the transporter (Huang et al., 2010).

| Co-administration of a mixture of herbal extracts increased the absorption of BGP-derived cinnamic acid derivatives in humans
To determine the effect of the herbal extract mixture on the absorption of the cinnamic acid derivatives of BGP in humans, plasma samples were collected for up to 24 h from participants who received BGP capsules or capsules containing a mixture of BGP and herbal extract powder.After enzymatic deconjugation, the plasma concentrations of artepillin C, drupanin, and their hydroxylated forms, capillartemisin A and 3,4-dihydroxy-5-prenylcinnamic acid, were analyzed by LC-MS/MS.Figure 1 shows the plasma concentration-time curves and AUC of these components.
When BGP and the herbal extracts were co-administered, the AUC values of artepillin C and drupanin were 312.9 ± 91.4 and 446.2 ± 144.5 ng/mL h, respectively; these values were 1.7-and 1.5-fold higher than those obtained when BGP was administered alone (p < .05for each).Furthermore, when BGP and the herbal extracts were co-administered, the AUC values of capillartemisin A and 3,4-dihydroxy-5-prenylcinnamic acid were 57.0 ± 16.5 and 34.5 ± 12.2 ng/mL h, respectively; these values were 1.6-and 1.8-fold higher than those obtained when BGP was administered alone (p < .05,p = .065,respectively).These results indicate that some components of the herbal extract mixture contribute to the increased absorption of the cinnamic acid derivatives of BGP.

| Turmeric extract enhanced the absorption of the cinnamic acid derivatives of BGP
To identify the herbal extracts that affect the absorption of cinnamic acid derivatives of BGP, rats were co-administered BGP and each herbal extract (turmeric, ginkgo leaf, coffee fruit, soybean, and gotu kola).Plasma samples were collected for up to 24 h after the ingestion of each mixture and subjected to LC-MS/MS analysis following enzymatic deconjugation.Figure 2a shows the AUC values for artepillin C, drupanin, capillartemisin A, and 3,4-dihydroxy-5-prenylcinnamic acid when each herbal extract was co-administered with BGP. (1) When BGP was co-administered with turmeric extract, the AUC value of artepillin C was 1377.2 ± 320.2 ng/mL h, which was significantly 1.4-fold higher than that when BGP was administered alone (1018.0 ± 184.5 ng/mL h, p < .05).In contrast, the co-administration of ginkgo leaf, coffee fruit, soybean, or gotu kola with BGP did not significantly alter the AUC values of artepillin C. When BGP was coadministered with turmeric extract, the AUC value of capillartemisin A was 365.7 ± 184.5 ng/mL h, a significant 1.7-fold increase compared F I G U R E 1 Plasma concentration-time curve and area under the curve (AUC) of the prenylated cinnamic acid derivatives in humans (mean ± SD; n = 6) within 0-24 h after ingestion of Brazilian green propolis (BGP) alone or a mixture of BGP and the herbal extracts.The plasma concentrations were analyzed using LC-MS/MS after enzymatic deconjugation.The AUC was calculated using the trapezoidal rule.*p < .05.Although no significant difference was found for drupanin when turmeric or gotu kola extract was co-administered with BGP, the AUC values were 626.9 ± 147.7 and 629.2 ± 166.8 ng/mL h, respectively.These values tended to increase 1.3-fold compared with those obtained when BGP was administered alone (p = .08each).The AUC values also tended to increase with 3,4-dihydroxy-5-prenylcinnamic acid (p = .08and .053,respectively).
F I G U R E 2 (a) Area under the curve (AUC) of the prenylated cinnamic acid derivatives when Brazilian green propolis (BGP) was coadministered with each herbal extract.The AUC was calculated using the trapezoidal rule.(b) Comparison of the plasma concentration-time curve of prenylated cinnamic acid derivatives in the BGP group (control) and the BGP + turmeric group.All data are expressed as mean ± SD, n = eight rats.*p < .05versus BGP (control).The plasma concentration-time curves of cinnamic acid derivatives after the co-administration of BGP and turmeric extract were compared with those after the administration of BGP alone (Figure 2b).The plasma concentration-time curve of artepillin C exhibited two distinct peaks at 30 min and 6 h after the coadministration of BGP and turmeric extract, and the concentrations were significantly increased by 1.7-and 1.5-fold, respectively, compared to those after the administration of BGP alone (p < .05).
Furthermore, the plasma concentrations of capillartemisin A at 30 min, 3 h, and 6 h after the co-administration of BGP and turmeric extract were 2.2-, 1.7-, and 3.0-fold higher than those after BGP ingestion, respectively (p < .05,for each).The plasma concentrationtime curves of drupanin and 3,4-dihydroxy-5-prenylcinnamic acid also revealed significant increases or increasing trends at 30 min and 3 h after the co-administration of BGP and turmeric extract compared to the ingestion of BGP alone.These results indicate that among the herbal extracts, turmeric extract increased the absorption of cinnamic acid derivatives in BGP.

| Cinnamic acid derivatives derived from BGP could be substrates for the drug elimination transporters
To determine whether artepillin C and drupanin are substrates for the drug elimination transporters P-gp and BCRP, their net flux ratios were calculated using an MDCK II cell monolayer overexpressing each transporter.Tables 2 and 3 show the results of the permeability studies using artepillin C and drupanin.The Papp (A to B) and Papp (B to A) values of artepillin C in MDCK II cells were 14.0 ± 0.7 and 11.2 ± 1.2 × 10 −6 cm/s, respectively, and the ER value was 0.8.In MDCK II-MDR1 cells overexpressing P-gp, the ER value of artepillin C was 0.9 and did not change with P-gp overexpression.Similarly, the ER values of drupanin in the MDCK II and MDCK II-MDR1 cell monolayers were 0.9 and 0.8, respectively (Table 2).
In the MDCK II-BCRP cell monolayer, the Papp (A to B) and Papp (B to A) values of artepillin C were 9.7 ± 0.2 and 16.0 ± 0.01 × 10 −6 cm/s, respectively, and those of drupanin were 1.3 ± 0.1 and 20.9 ± 0.1 × 10 −6 cm/s, respectively.The ER values of artepillin C and drupanin in cells overexpressing BCRP were 1.6 and 15.7, respectively, whereas those in the parental cell line were 0.8 each.The net flux ratios of artepillin C and drupanin were 2.0 and 20.4,respectively, suggesting that BCRP mediated the efflux of artepillin C and drupanin as substrates (Table 3).

| DISCUSS ION
In this study, the co-administration of turmeric extract with BGP increased the absorption of prenylated cinnamic acid derivatives of BGP.Moreover, artepillin C and drupanin, the main cinnamic acid derivatives of BGP, were confirmed to be substrates of the drug elimination transporter, BCRP.
The turmeric extract was standardized to contain 70% or more curcumin.Curcumin has been reported to inhibit P-gp and BCRP, which are drug elimination transporters (Lee et al., 2011;Zou et al., 2020), and affect the absorption and excretion of several compounds that act as P-gp and/or BCRP substrates (Ge et al., 2016;Tong et al., 2023;Zhou et al., 2015).For example, the uptake of sulfasalazine increases in mice when co-administered with 300-400 mg/kg curcumin (Kusuhara et al., 2012;Shukla et al., 2009).In this study, the plasma concentration-time curves of the cinnamic acid derivatives showed two peaks at 30 min and 3-6 h after the co-administration of BGP and 200 mg/kg curcumin in the turmeric extract; these values were significantly higher than those obtained when BGP was administered alone (Figure 2b).As compounds with two peaks in the plasma concentration-time curves are considered to be the result of enterohepatic circulation (Yan et al., 2006), the first absorption phase after oral ingestion of the cinnamic acid derivatives and the intestinal reabsorption phase of the cinnamic acid derivatives excreted in the bile would increase because curcumin inhibits their transport to the intestine via BCRP.The elevated uptake of cinnamic acid derivatives in the body can enhance the pharmacological activities of BGP.For instance, when combined with herbal extracts, BGP is effective in improving cognitive function in elderly people, even at lower doses than when administered alone (Asama et al., 2020(Asama et al., , 2021)).
In addition, artepillin C increases mitochondrial UCP1 expression and induces the formation of beige adipocytes in the white adipose tissue of mice (Nishikawa et al., 2020).Curcumin also enhances the induction of beige adipocyte formation by artepillin C (Nishikawa et al., 2019).Although the actual plasma artepillin C concentration was not confirmed in these studies, an increase in the plasma artepillin C concentration due to curcumin inhibition by BCRP may be one of the factors contributing to the synergistic effect.
BCRP expression is not restricted to the intestine but extends to the liver and kidney and is known to contribute to the urinary and biliary excretion of substrates (Mizuno & Sugiyama, 2002), leading to substrate accumulation in the body.In the present study, the plasma concentrations of artepillin C and drupanin did not differ at 24 h post-ingestion between the rats administered BGP alone and those administered BGP and turmeric, suggesting that the effect of curcumin on the clearance of these compounds was less than that on their absorption (Figure 2b).This result could be related to the metabolism of curcumin in vivo.Curcumin is metabolized to glucuronide and sulfate conjugates, and the IC50 values of these conjugates for BCRP are higher than those of curcumin (Heger et al., 2013;Karibe et al., 2018).Although the amount of curcumin and its metabolites distributed in the liver and kidneys should be verified because of the dose of curcumin used, curcumin may not have reached these organs in sufficient amounts to inhibit BCRP.
In the bidirectional transport assay using MDCK II-BCRP cells, the ER value of drupanin was 15.7, which was approximately 10fold higher than that of artepillin C (Table 3).These results suggested that the affinity of drupanin for BCRP was higher than that of artepillin C.However, the increases in the AUC values of artepillin C and drupanin in humans after co-administration of BGP and the herbal extract mixture were 1.7-and 1.5-fold, respectively, compared to that of BGP administration alone (Figure 1).Similarly, in rats, the AUC values of artepillin C and drupanin increased 1.4-and 1.3-fold, respectively, owing to the co-administration of BGP and turmeric extract (Figure 2a), suggesting no difference between the absorption of artepillin C and drupanin due to the co-administration of BGP and turmeric extract in humans and rats.The difference between the in vitro and in vivo results suggests that the affinities of artepillin C and drupanin for BCRP are altered by metabolism in the gastrointestinal epithelium.Our previous studies have revealed that artepillin C and drupanin are absorbed into the body not only as aglycones but also as conjugation metabolites in humans and rats.
Furthermore, artepillin C is mainly metabolized to glucuronide in both humans and rats, whereas drupanin metabolites are chiefly glucuronide in humans and sulfate in rats (Yamaga et al., 2021(Yamaga et al., , 2022)).Among the flavonoids, genistein has been reported to be excreted as a BCRP substrate following its metabolism to glucuronide or sulfate conjugates (Zhu et al., 2010).Ko-143, a BCRP inhibitor, causes greater inhibition of the efflux of chrysin sulfate to the outside of cells than glucuronide, suggesting that chrysin sulfate has a higher affinity for BCRP than chrysin glucuronide (Ge et al., 2015).Therefore, the affinity of substrates for BCRP could be altered by conjugation metabolism; however, the pattern of changes in the binding affinity for BCRP is substrate-dependent and not specifically defined.To understand the effect of BCRP on the absorption of artepillin C and drupanin, differences in the affinities of their conjugated metabolites for BCRP compared to aglycone had to be confirmed.
Generally, the absorption and metabolism of active constituents in foods and plants are influenced by the presence of other foods and herbal constituents (Colalto, 2010;Sun et al., 2019).
However, the co-administration of ginkgo leaf, coffee fruit, or soybean did not induce significant changes in the absorption of BGP components in rats (Figure 2a).In mice, the AUC and C max of sulfasalazine were not altered by the co-administration of a single dose of ginkgo leaves and sulfasalazine, whereas the oral administration of ginkgo leaf extract for 5 days significantly suppressed the intestinal BCRP expression levels and increased the AUC and C max of sulfasalazine by twofold compared with the administration of ginkgo leaves alone (Kim et al., 2021).Similarly, the administration of 80 mg/kg Ginkgo leaf extract for 10 days was reported to increase the AUC and C max of atorvastatin, a BCRP substrate, in rats (Ren et al., 2019).Therefore, repeated administration of a mixture of BGP and herbal extracts may further increase the absorption of cinnamic acid derivatives compared with a single administration.A mixture of flavonoids and soybean-derived phosphatidylcholine was found to improve the absorption of flavonoids via physical treatment to form liposomes (Wang et al., 2015).Therefore, the absorption of cinnamic acid derivatives can be improved not only by co-administration, as observed in the present study, but also by liposome formation with phospholipids.Contrarily, the AUC values of drupanin and 3,4-dihydroxy-5-prenylcinnamic acid tended to increase with the co-administration of gotu kola extract and BGP (Figure 2a, p = .08and .053,respectively).Gotu kola contains asiaticoside, a saponin, as the characteristic component (Idris & Nadzir, 2021;Paine, 2020).Saponins, which are amphipathic compounds with surface activity (Kunjumon et al., 2022), may improve the membrane permeability of drupanin.The AUC of artepillin C was not changed by the co-administration of gotu kola extract and BGP.
Therefore, further detailed studies could shed light on the relationship between the differences in the structures and absorption processes of cinnamic acid derivatives.

| CON CLUS ION
To the best of our knowledge, this is the first study to demonstrate

ACK N OWLED G M ENTS
None.

CO N FLI C T O F I NTE R E S T S TATE M E NT
M.Y., H.K., H.T., and A.Y. are employees of Yamada Bee Company, Inc., a company engaged in research and commercial endeavors related to bee products.The authors declare no competing financial interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data underlying this study can be obtained from the corresponding author upon request.

E TH I C A L S TATEM ENT
The protocol for this clinical trial was approved by Yamada Bee Co.
Ethical Committee.The study was conducted in accordance with the ethical principles of the Declaration of Helsinki (approval no. with that obtained when BGP was administered alone (p < .05).
2020015).This animal study was approved by the Animal ResearchEthics Committee of KAC Co., Ltd.(approval No. 22-1217).All rats were handled in accordance with the Animal Care and Use Regulations of KAC Co. Ltd.I N FO R M E D CO N S E NTAll participants were informed of the research protocol and they have signed an informed consent form.